CA2106167A1 - Formulation for stabilizing enzymatic activity and immunoreactivity of creatine kinase and creatine kinase isoenzymes - Google Patents
Formulation for stabilizing enzymatic activity and immunoreactivity of creatine kinase and creatine kinase isoenzymesInfo
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- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/50—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving creatine phosphokinase
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Abstract
TITLE
FORMULATION FOR STABILIZING ENZYMATIC ACTIVITY AND
IMMUNOREACTIVITY OF CREATINE KINASE AND CREATINE
KINASE ISOENZYMES
ABSTRACT OF THE DISCLOSURE
A formulation for stabilizing enzymatic activity and immunoreactivity of creatine kinase and creatine kinase isoenzymes comprising animal serum, creatine kinase and/or creatine kinase isoenzymes, an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7?0.1, an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or creatine kinase isoenzyme thiol groups, an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatine kinase isoenzymes, and an effective amount of an antimicrobial to prevent microbial growth is provided.
FORMULATION FOR STABILIZING ENZYMATIC ACTIVITY AND
IMMUNOREACTIVITY OF CREATINE KINASE AND CREATINE
KINASE ISOENZYMES
ABSTRACT OF THE DISCLOSURE
A formulation for stabilizing enzymatic activity and immunoreactivity of creatine kinase and creatine kinase isoenzymes comprising animal serum, creatine kinase and/or creatine kinase isoenzymes, an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7?0.1, an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or creatine kinase isoenzyme thiol groups, an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatine kinase isoenzymes, and an effective amount of an antimicrobial to prevent microbial growth is provided.
Description
211 ~61~7 TITL_E
S FORMULATION FOR STABLIZ~G ENZYMAIIC ACTIVITY AND
IMMUNOREACIIVITY OP CREAlINE KINASE AND (: REATINE
KINASE ISOE~Y~
TEÇHNT~, FIELD
This invention relates to a foImulation for stabilizing the enzymatic activity and immunoreactivity of creatine kinase and cr~atine kinase isoenzymes contained in human serum samples and in reconstitutable lyophilized con~rol products used for the determination of creatine kinase or creatine kinase S isoenzyme levels in human serum samples.
BA~KGR5)~ ART
The determination of creatine kinase (CK) or CK
isoenzyme levels ïn human serum is important in diagnosing a number of physiological conditions. An increased level of CK
S FORMULATION FOR STABLIZ~G ENZYMAIIC ACTIVITY AND
IMMUNOREACIIVITY OP CREAlINE KINASE AND (: REATINE
KINASE ISOE~Y~
TEÇHNT~, FIELD
This invention relates to a foImulation for stabilizing the enzymatic activity and immunoreactivity of creatine kinase and cr~atine kinase isoenzymes contained in human serum samples and in reconstitutable lyophilized con~rol products used for the determination of creatine kinase or creatine kinase S isoenzyme levels in human serum samples.
BA~KGR5)~ ART
The determination of creatine kinase (CK) or CK
isoenzyme levels ïn human serum is important in diagnosing a number of physiological conditions. An increased level of CK
2 0 can be indicative of myocardial infarction, myocardial ischemia, stenocardia, ,tachycardia, myocarditis, subarachnoid hemorrage, stroke, brain tumor, meningitis and encephalitis, among other conditions.
Three predominant isoenzymes of CK are recognized;
2~ ~hese are dimers consisting of the M and B sub-units. The predominant dimer presen~ in the blood, serum or plasma of normal individuals is CK-MM isoenzyme, with variable but usually trace quantities of CK-MB that indicate the normal degradation of skeletal muscle. The CK-BB isoenzyme is not 3 0 usually present in detectable amounts in serum of normal individuals but is present in significant quantities in brain tissue and smooth rnuscle tissue.
Elevations of the CK-BB isoenzyme can occur in physiological conditions such as metastatic carcinoma or severe 35 burns. Elevated levels of CK-MB isoenzyme have been used as an indicàtor of myocardial infarction where possible sources of 2 ~ 7 significant skeletal muscle damage can be eliminated. More particularly, repetitive determinations of CK-MB levels in serum can indicate the time course and severity of infarctions.
More recen~ly, the isoforms of CK isoenzymes have S received attention as pote~tial markers for early diagnosis of myocardial infarction. There are three isoforms of CK-MM
isoenzyme; CK-MM1, CK-MM2 and CK-MM3; and two isoforms of CK-MB isoenzyme; CK-MB1 and CK-MB2. Shortly after myoca~dial infarction, the ratio of CK-MM and ~K-MB isoforms contained in tissue to that contained in plasma increases. Such increase occurs several hours before either ~otal CK or CK-MB
activity exceeds normal reference values.
A number of diagnostic assays have been developed for the de~ermination of the presence or the concentration of CK or CK isoen~ymes in human serum. Many such assays are based on measuremen~ of the enzymatic activity of the CK enzyme or isoenzyme. Difficulties in measuring the en7ymatic activity of the CK-MB isoenzyme and the need for greater sensitivity have } led to the developmen~ of immunochemical mass determinations for CK-MB. Such assays are highly dependent on the immunoreactivity of the CK-MB isoenzyme.
- It has been observed ~hat CK and CK isoenzymes are highly unstable. CK and CK isoenzymes are sulfhydryl-requiring, that is, they need to maintain their -SH groups in 2 5 their free, unoxidized form. Atmospheric oxygen causes the oxidation of the -SH groups to the disulfide linkage, thereby diminishing en~yme stability and catalytic activity.
Additionally, changes to the CK and CK isoenzyme molecules caused by increased temperatures and the presence of 3 0 proteases and reducing agents can change ~he immunoreactivity of the molecules.
Storage stability of patien~ serum samples containing CK
and CK isoenzymes is important, especially when further analysis on a sample is requested retrospectively following an 3 5 equivocal ECG or when the cause of a raised total CK level is `
~. ~ 3 ~ '7 i being sought. Such a request nnay be made the same day or days after ~he initial ECG or CK assay was performed. Buttery et al. [Clinical Biochemistry, 2S~ 13 (1992)] report that samples stored at -20C overnight show, on average, a decrease S in CK-MB isoenzyme values of 13.5%. Such a decrease could result in a misdiagnosis. Prolollged storage of samples at room temperature for 4-6 days showed a 50% deterioration of CK-MB
when measured electrophoretically and 20% deterioration when measured by an immunochemical mass assay. Therefore, 10 a need exists for a means for stabilizing CK and CK isoenzyme activity in patient serum samples.
CK and CK isoenzyme assay control products containing CK
or CK isoenzyme also suffer from instability problems. In order ` to facilitate storage of such control products, they are typically 15 Iyophilized. A useful Iyophilized CK or CK isoenzyme control product must have two characteristics which are often very difficult to achieve simultaneously. First, there must be substantially no or only very small temperature of hydration effect and, second, there mllst only be very slight changes in 2 0 enzyme activity or immunoreactivity upon standing after reconstitution (rehydration) of the lyophilized control product.
The temperature of hydra~ion effect is the phenomenon of variability of recovered enzyme activity and immunoreactivity from a Iyophilized control product upon rehydration caused by 2 5 the differences in the temperature of water used. For practical purposes, for use in diagnostic assays, lyophilized control products must have a small temperature of hydration effect to permit reproducible analytical results.
Thiols, sucb as N-acetyl cysteine, glutathione and 30 monothioglycerol, have been used as additives to lyophilized CK
and CK isoenzyme control products as thiol protectors. (See U.S.
Patent No. 4,442,212, issued April lû, 1984 to Briggs~) While use of such thiols in lyophilized CK and CK isoenzyme control products has been found to control the stability of CK and CK
3 5 isoenzyme enzymatic activity after reconstitution, use of thiols p~
. 4 has lit~le effect on stability of CK and CK isoenzyme immunoreactivity.
U.S. Patent No. 4,994,375, issued February 19, 1991 to Posner et al., discloses a stable reconstituted human serum-based control for the assay of total lactate dehydro~enase (LDH) and CK and their isoenzymes which is comprised of human serum, LDH and LDH isoenzymes, CK and CK isoenzymes and sodium citrate. The pH of the control product is adjusted to 6.7 before Iyophilization. In this contlol product formulation, there is no means disclosed for maintaining the pH
of the control product at 6.7 after rehydration. Variability in pH of the con$rol product after reforrnulation can lead to inconsistent results. Additionally, there is no suggestion that the disclosed control product formulation would stabilize immunoreactivity of CK or CK isoenzymes.
There is a need for a formulation for stabilizing enzymatic activity and immunore~ctivity of CK and CK
isoenzymes which can be used to stabilize h-~man serum samples containing CK and CK isoenzymes and as a reconstitutable lyophilized CK and CK isoenzyme control product which exhibits little temperature of hydration effect and which is stable after reconstitution.
D~S~L~ lN~Nl~ON
The formulation of this invention for stabilizing 2 5 enzymatic activity and immunoreactivity of creatine kinase or creatine kinase isoenzymes comprising:
(a) animal serum;
~b) creatine kinase and/or creatine kinase isoenzymes;
3 0 (c) an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7 0.1;
(d) an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or crea~ine kinase 3 5 isoenzyme thiol groups;
, ` . 2 ~ 7 (e~ an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatin~
kinase isoenzymes; and (f) an effective amount of an antimicrobial to 5 prevent microbial growth in the ~ormulation.
The Iormulation oE ~he in~stant invention can be used to s~abilize enzymatic activity and ;mmunoreactivity of CK and CK
isoenzymes in human serum samples and in reconstitutable lyophilized control products for the assay of CK or CK-MB in 10 human serum. When used as a reconstitutable lyophilized control product, the formulation of the instant invention exhibits little temperature of hydration effect providing a cvntrol product in which enzymatic activity and immunoreactivi~y remain constant after reconstitution for at 15 least two weeks at 4C.
D:EiSC~le~
The formulation of the instant invention is useful for stabilizing both the enzymatic activity and immunoreactivity of CK and CK isoenzymes in human serum samples and in 2 0 recons~ituted Iyophilized CK and CK isoenzyme control products.
The formulation can take the form of an additive to blood collection tubes for the collection of human serum samples to be assayed for the dete~mination of CK and CK isoenzyme levels or ~he formula~ion can ~ake the form of a reconstitutable 25 lyophilized control product to be used in an assay for the determination of CK andlor CK isoenzyme levels.
The formulation of the instant invention is serum-based.
Where the formulation is used in blood collection tubes to preserve the stability of CK and CK isoenzymes in human serum 3 0 samples, the serum is that collected as the sample. Where the formulation talces the form of a reconstitutable Iyophilized CK
and/or CK ;soenzyme control product, the serum can be any animal serum. Horse serum is preferred.
When the formulation is used as a control product, i~ is 3 5 preferred that the serum have substantially low or no residual "
}~ 6 2 1 ~
CK and CK isoenzyme enzymatic activity or immunoreactivity.
~, Residual enzymatic activity and immunoreactivity can be removed from the serum by heat treatment of the serum. The serum can be heated to any temperature for a length of time S which will denature the CK or CK isoenzyme, such as S6C for one hour or 60C for one minute. Heat treatment of the serum for one minute at 60C is preferred.
CK and CK isoenzymes can be ~ound in the human serum samples collected for assaying for CK and CK isoenzymes or, if 10 the formulation is used as a reconstitutable lyophilized control product, CK and CK isoenzymes c~n be added to the serum base.
If the serum base of the formulation is treated to destroy residual CK and CK isoenzyme enzymatic activity and irnmunoreactivity, the CK and CK isoenzyme should be added to 15 the serum base after such treatment has occurred. The reconstitutable control product can be a control product for the assay of both CK and CK isoen~ymes, in which case, both (:K and CK isoenzymes would be present in the formulation Alternatively, the control product can be used for the assay of 2 0 only CK or a CK isoenzyme, in which case, only CK or the CK
isoenzyme would be present in the formulation. CK or CK
isoenzyme from any animal muscle or heart tissue, such as cynamolgus monkey, rabbi~ and human, can be used when the con~rol product is to be used in conjunction with a diagnostic 2 5 assay which measures the enzymatic activi~y of the CK enzyme or isoenzyme When the diagnostic assay is an immunochemical assay, animal CK or CK isoenzyme which cross-reacts with antibody to human CK or CK isoenzyme can be used Purified human CK or CK isoenzyme is preferred, 30 regardless of the type of diagnostic assay with which the control product is to be used.
One of the key elements of the formulation of the instant invention is that the formulation pH is maintained at 6.7 i 0.1, even after reconstitution of the Iyophilized control product.
3 5 Maintenance of the formulatioll pHI is achieved by including a 7 ~
2~6~7 t buffer having a pKa in the range of 6.2-7.6. Suitable buffers include piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-2-acetamidoiminodiacetic acid (ADA), 1,3-bis-[~ris(hydroxymethyl)methylamino] propane (BIS-TRIS
S PROPANE), N-2-acetamido-2-aminoethanesulfonic acid (ACES), imidazole, diethylmalonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acld (HEPES), and phosphate. PIPES buffer is preferred. An effective amount of buffer is the concentration of buffer which is needed to maintain pH of the formulation 10 between 6.6 and 6.8. A PIPES bufiFer concentration of 80-95 mM has been shown to be effectiYe.
' As described above, in order to stabilize CK and CK
isoenzyme enzymatic activity and immunoreactivity, it is important to prevent oxidation of the CK and CK isoenyzme 15 thiol groups. The formulation of the instant invention does so by containing a non-thiol antioxidant, such as ascorbic acid, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Ascorbic acid is preferred. An effective amount of non--thiol antioxidant is the concentration of antioxidant needed to 2 0 prevent oxidation of the thiol groups. Concentrations of ascorbic acid in the range of 10-100 mM have been shown to be effective, 30 mM being preferred Surprisingly, it was found that inclusion of a non-reducing polyol into the formulation conferred greater stability 2~ of CK and CK isoenzyme immunoreactivity. Any non-reducing polyol, such as sucrose, glycerol3 trehalose and mannitol, can be used in the formulation of the instant invention. Sucrose is preferred. An effective amount of non-reducing polyol is the concentration of polyol which is needed to stabilize ~he 3 0 immunoreactivity of CK and CK isoenzyme. A concentration of non-reducing polyol of 1% or greater has been ~ollnd to be effective.
Microbial growth can produce pro~eases and other enzymes which can destroy both enzymatic and antigenic 3 5 activity of CK and CK isoenzymes. Therefore, an antimicrobial 8 2 ~
îs included ;n the formulation of the instant invention to prevent microbial growth. Any aminoglycoside antimicrobial, such as gentamicin, streptomycin, neomycin and kanamycin, can be used. &entamicin is preferred. An effective amount of S aDtimicrobial is the concentration of antimicrobial needed to prevent microbial growth. A 0.1% concentration of gentamicin has been found to be effective.
The formulation of the instant invention can be used to stabilize human selum samples collected in blood collection tubes. Buffer, non-thiol antioxidant, non-reducing polyol and antimicrobial can be added in dry form to blood collection tubes for use in collec~ing the human serum samples containing CK and CK isoenzymes.
Alternatively, the formulation can be used as a l S reconstitutable lyophilized control product. All of the components, except CK and CK isoenzyme, can be added in any order to the serum base of the formulation. The formulation can then be pH adjusted to 6.7i0.05, sterile filtered and heat ~eated, if so desired. CK and CK isoenzyme must be added after pH adjustment and heat treatment so as to avoid destruction of CK and CK isoenzyme. The serum-based formulation can then be lyophilized by any standard Iyophilization technique. Since the lyophilized control product is stable, it can be reconstituted with water and used in - 2 5 conjunction with, for example, automated clinical analyzers - utilized for the measurement of CK and/or (:K isoenzymes.
Once reconstituted, the formulation of the instant inverltion has been found to remain stable for at least two weeks when stored at 4C.
The following examples illustrate the invention.
~-- 9 EiXAMPLES
E~AMPLE I - PREPAR~TIQ~ OF lRECQN~T[IUT~BLE
s L~ P~E2 MB LD~R~A~
I. Preparation of CK-MB Calibrator Matrix - To 23 liters of horse serum (Pel-freez Biologicals) was 10 added 616.4 gm of 80 mM PIPES 1.5 Na salt (Sigma Corp.~, 23 gm of gentamicin sulfate (Sigma Corp.3, 230 gm of sucrose ~Ul~a Pure) and 121.6 gm ascorbic acid (Calbiochem Corp.) with continuous mixing until all additives were completely dissolved in the horse serum. While stirring vigorously, but 15 not so as to cause foaming, the horse serum mixture was adjusted to a pH of 6.7iO.05 at room temperature by slowly adding sodium hydroxide.
After pH adjustment, the horse serum mixture was filtered through a 0.2-mieron filter into a heat treatment tank 20 containing a submersible heating coil. The horse serum mixture was heat treated to remove residual CK and CK
isoenzyme by heating the mixture to SO~C for one minute.
Horse serum containing ~he above-identified additives, adjusted to pH 6.7iO.05, filtered and heat treated is CK-MB
25 Calibrator Matrix. The Matrix was eovered and stored refrigerated for further use.
II. Preparation of CK-MB Stock Solution A lyophilized preparation of CK-MB (Lee Scientific~ was 3 0 used as the source of CK isoenzyme. The lyophilized S:K-MB
preparation was rehydra~ed with 16 mL of (: alibrator Matrix to prepare CK-MB S~ock Solution. To three lO0 mL volumetric flasks was added 25 ~,IL of Stock Solution and enough Matrix to bring the resulting solution to 100 mL. These solutions were 35 used as samples on the acat~plws immunoassay system (E~. I. du Pont de Nemours and Company) and analyzed for mass CK-MB
content using a mass immunochemical assay. Results of this assay showed that the CK-MB S~ock Solution had a CK-MB
concentra~ion of 383,000 ,ug/L
S III. Formulation of CK-MB Calibrator Levels 1, 2, Low, Medium, High and 3 Using the CK-MB concentration of CK-MB Stock Solution determined in Section II., above, it was calculated, using a mass balance equation, that 9.9 gm of (:K-MB Stock Solution 10 needed to be added ~o 9070 gm of A~atrix to bring the Matrix to a target CK-MB concentration of 420 ~,lg/I,. The Matrix was assayed on the aca~plus immunoassay system to determine CK-MB concentration. 'rhe result was lower ~han the expected 420 llg/L and an additional 2.3 gm of CK-MB Stoclc Solution 15 was added to the Ma~ix. This was de3ignated Calibrator Level 3. Calibrator Matrix alone was used as Calibrator Level 1, containing 0 llg/mL CK-MB. Calibrator Levels High (200 ~glmL), Medium (lOO,ug/mL), Low (50 ~lg/mL) and 2 (20 ,ug/mL) were prepared by mixing together 1414 gm Matrix 20 and 1286 gm Level 3, 2057 gm Ma~rix arld 643 gm Level 3, 2379 gm Ma~rix and 321 gm Level 3 and 6200 gm Matrix with 310 gm Level 3, respectively.
For each level of CK-MB Calibrator, Iyophilization vials were filled with 5.39 gm of calibrator and topped with half-5 inserted slotted s~oppers. The vials were Iyophilized accordingto a routine protocol. The vials were frozen to apprcximately -38C. When the condenser reached approximately -80C, the chamber was evacuated to 200 millitorr and the shelf tempera~ure was gradually increased to 30C over 30 approximately 45 hours. The chamber was then held at 30C
and 30 millitolT for ten hours. The Iyophilized vials were stoppered under a vacuum of approxima~ely 30 millitorr and cooled to 4C.
2 ~
EXA~MPLE II - ~TAlBlI,Il~ ~;; OF RE~C)NSTIT5~ATED CK-MB
3RATC~RS
One vial each of lyophilized CK-MB Calibrator Levels 1, 2, Low, Medium, High and 3, prel)ared as described ;n Example I, 5 were reconstituted with 5.0 mI, of room temperature water on each of Days 0, 8, 22 and 44. The reconstituted vials prepared on Days 1, 8 and 22 were storecl at 4C until Day 44. Cal;brator reconstituted on Day 44 served as a control as it contained the original amount of CK-MB in each calibrator level. On Day 44, 10 three replicates of each level of reconstituted CK-MB Calibrator from each day of reconsti~ution were assayed on the aca~plus immunoassay system to determine the amount of CK-MB in each CK-MB calibrator level using a mass immunochemisal CK-MB method of determination. M[eans of the replicates, standard 15 deviations and percent coefficients of variation were calculated.
Using the slope and intercept calculated ~rom a linear least squares correlation for each level, a percent loss per day was calculated. Prom the percent loss per day was calcula~ed the number of days from reconstitution undl a 5% loss of CK-MB
2 0 would result for each reconstituted calibrator level. Results are depicted in Table 1, below.
_ l 2 2 ~ 7 ~` ~G9~]3LE 1 i DAYS FROM MEAN NU~IBEROF
RECONSTTrL~ON CK-~EI% LOSS DAYS FOR
5 LE~3L 5~EEIlESI~i 31 LkL~aC,y. P~R DAY 5% LOSS
1 0 3q.15 1.29 3.79 1 B 34.03 0.84 2.46 1 22 34.67 0.41 1.17 1 0 1 44 34.75 1.13 3.25 i 0 63.28 0.2S 0.39 0.06 77 2 8 64.02 0.93 1.45 2 22 65.07 0.96 1.47 1 5 2 44 64.18 0.10 0.16 !
LOW 0 112.07 0.70 Q.63 0.15 34 ~OW 8 1~0.07 2.85 2.59 LOW 22 115.23 4.22 3.66 2 0 LOW 44 116.10 3.90 3.36 ~DEDIUM 0 192.35 4.17 2.17 0.22 23 ~DEDIUM 8 202.40 5.09 2.52 MEDIlUM 22 207.07 6.49 3.14 2 5 MEDIUM 44 211.23 3.85 1.82 HIGH 0 388.95 9.97 2.S6 0.19 26 H~ 8 396.63 1.76 0.44 H~GH 22 406.80 2.26 0.56 3 0 HIGH 44 421.65 11.95 2.83 3 0 850.93 5.39 0.63 0.08 64 3 8 888.40 12.30 1.38 3 22 8~0.65 g.69 1.10 3 ~ 3 44 891.5S 3.18 0.3fi .
Results shown in Table 1, above, indicate that the reconstitutable lyophilized CK-MB CalibTator of the instant invention will not lose more than 5% of the original amount of 5 CK-MB contained in the calibrator for at leas~ 23 days after reconstitution when stored at 4~C.
EXAMPLE III - l~e9F ~IYDRAI'IO~ EFFEÇ E
Three vials for each of Levels 2 and 3 of the CK-MB
Calibrato~ of the instan~ invention, prepared according to Example I, above, were rehydrated wi~h water; one vial for each of water temperatures of 4, 25 and 37C. Also rehydrated at 4, ~5 and 37C were samples of lyophilized 15 stripped human serum at low and high levels of CK-MB, prepared according to U. S. Patent No. 4,264,471, issued to Briggs on April 28, 1981, and commerical CK-MB calibrators and controls from the Tandem~-E CKMB II ImmunoEnzMetric Assay ~Hybritech, Inc.), the Stratus~) CK-MB Fluorometric 20 Enzyme Immunoassay Kit (Baxter Diagnostics, Inc., Dade Division~ and Cala. The reconstituted calibrators and controls were assayed in triplicate for the amount of CK-MB contained therein using the aca@~plus immunoassay system and a mass immunochemical method of determination. Mean CK-MB
2 5 values were determined for each sample and the percent change in CK-MB value fiom that of the 4C rehydrated sample was calculated: Results are depicted in Table 2, below.
i. 1 4 --A~i 2 1 ~ 6 I ~ 7 `#
L~ 2 ~:k~ % LOS~ OF CK-MB
SAM~ ~ 2 ~ 37 INVENTION, LEVEL 2 1~.2 14.9 13.8 2.0 9.2 INVENTION, LE~VEL 3 340.0 324.0 318.0 4.7 6.5 STRIPPED HUMAN
SERllM, LOW 14.6 10.9 8.0 2S.3 4~.2 STRIPPED HUMAN
SERUM, HIGH ~68.0 222.0 170.0 17.2 36.6 1 5 TANDEM~)-E CONTROL, LOW 15.1 14.0 14.1 7.3 6.6 TANDEM~)-E CONT~OL, HIGH 56.3 52.7 50.8 6.4 9.8 2 0 TANDEM~)-E CALIBRATOR, HI&H 35.7 36.3 33.3 -1.7 6.7 STRATUS~) CALIBRATOR, MID 16.9 13.4 10.4 20.7 38.5 g STRATUS~9 CALIBRATOR, HIGH 37.9 30.0 21.7 20.8 42.7 CALA, PSID 25.8 25.3 25.1 1.9 2.7 3 0 CALA,HIGH 60.4 58.9 57.4 2.5 5.0 As can be seen from Table 2, the reconstitutable lyophilized CK-MB control product of the instant invention exhibit little temperature of hydration effect. It is desirable 3 ~ that the ternperature of hydration effect be as small as possible Only the Cala controls exhibited lower temperature of hydration efPect than did the calibrators of the instant invention. The Tandem~)-E calibrator and controls performed ~- connparably to the instant invention at 37C, but worse at 25C.The Stratus(~) and stripped hunnan serum calibrators showed significantly greater temperature of hydration effect than did the reconstitutable lyophilized CK-MB control product of the S instant invention. The results i~or stripped serum show the magnitude of temperature of rehydration effect one could expect without the instant invention.
Three predominant isoenzymes of CK are recognized;
2~ ~hese are dimers consisting of the M and B sub-units. The predominant dimer presen~ in the blood, serum or plasma of normal individuals is CK-MM isoenzyme, with variable but usually trace quantities of CK-MB that indicate the normal degradation of skeletal muscle. The CK-BB isoenzyme is not 3 0 usually present in detectable amounts in serum of normal individuals but is present in significant quantities in brain tissue and smooth rnuscle tissue.
Elevations of the CK-BB isoenzyme can occur in physiological conditions such as metastatic carcinoma or severe 35 burns. Elevated levels of CK-MB isoenzyme have been used as an indicàtor of myocardial infarction where possible sources of 2 ~ 7 significant skeletal muscle damage can be eliminated. More particularly, repetitive determinations of CK-MB levels in serum can indicate the time course and severity of infarctions.
More recen~ly, the isoforms of CK isoenzymes have S received attention as pote~tial markers for early diagnosis of myocardial infarction. There are three isoforms of CK-MM
isoenzyme; CK-MM1, CK-MM2 and CK-MM3; and two isoforms of CK-MB isoenzyme; CK-MB1 and CK-MB2. Shortly after myoca~dial infarction, the ratio of CK-MM and ~K-MB isoforms contained in tissue to that contained in plasma increases. Such increase occurs several hours before either ~otal CK or CK-MB
activity exceeds normal reference values.
A number of diagnostic assays have been developed for the de~ermination of the presence or the concentration of CK or CK isoen~ymes in human serum. Many such assays are based on measuremen~ of the enzymatic activity of the CK enzyme or isoenzyme. Difficulties in measuring the en7ymatic activity of the CK-MB isoenzyme and the need for greater sensitivity have } led to the developmen~ of immunochemical mass determinations for CK-MB. Such assays are highly dependent on the immunoreactivity of the CK-MB isoenzyme.
- It has been observed ~hat CK and CK isoenzymes are highly unstable. CK and CK isoenzymes are sulfhydryl-requiring, that is, they need to maintain their -SH groups in 2 5 their free, unoxidized form. Atmospheric oxygen causes the oxidation of the -SH groups to the disulfide linkage, thereby diminishing en~yme stability and catalytic activity.
Additionally, changes to the CK and CK isoenzyme molecules caused by increased temperatures and the presence of 3 0 proteases and reducing agents can change ~he immunoreactivity of the molecules.
Storage stability of patien~ serum samples containing CK
and CK isoenzymes is important, especially when further analysis on a sample is requested retrospectively following an 3 5 equivocal ECG or when the cause of a raised total CK level is `
~. ~ 3 ~ '7 i being sought. Such a request nnay be made the same day or days after ~he initial ECG or CK assay was performed. Buttery et al. [Clinical Biochemistry, 2S~ 13 (1992)] report that samples stored at -20C overnight show, on average, a decrease S in CK-MB isoenzyme values of 13.5%. Such a decrease could result in a misdiagnosis. Prolollged storage of samples at room temperature for 4-6 days showed a 50% deterioration of CK-MB
when measured electrophoretically and 20% deterioration when measured by an immunochemical mass assay. Therefore, 10 a need exists for a means for stabilizing CK and CK isoenzyme activity in patient serum samples.
CK and CK isoenzyme assay control products containing CK
or CK isoenzyme also suffer from instability problems. In order ` to facilitate storage of such control products, they are typically 15 Iyophilized. A useful Iyophilized CK or CK isoenzyme control product must have two characteristics which are often very difficult to achieve simultaneously. First, there must be substantially no or only very small temperature of hydration effect and, second, there mllst only be very slight changes in 2 0 enzyme activity or immunoreactivity upon standing after reconstitution (rehydration) of the lyophilized control product.
The temperature of hydra~ion effect is the phenomenon of variability of recovered enzyme activity and immunoreactivity from a Iyophilized control product upon rehydration caused by 2 5 the differences in the temperature of water used. For practical purposes, for use in diagnostic assays, lyophilized control products must have a small temperature of hydration effect to permit reproducible analytical results.
Thiols, sucb as N-acetyl cysteine, glutathione and 30 monothioglycerol, have been used as additives to lyophilized CK
and CK isoenzyme control products as thiol protectors. (See U.S.
Patent No. 4,442,212, issued April lû, 1984 to Briggs~) While use of such thiols in lyophilized CK and CK isoenzyme control products has been found to control the stability of CK and CK
3 5 isoenzyme enzymatic activity after reconstitution, use of thiols p~
. 4 has lit~le effect on stability of CK and CK isoenzyme immunoreactivity.
U.S. Patent No. 4,994,375, issued February 19, 1991 to Posner et al., discloses a stable reconstituted human serum-based control for the assay of total lactate dehydro~enase (LDH) and CK and their isoenzymes which is comprised of human serum, LDH and LDH isoenzymes, CK and CK isoenzymes and sodium citrate. The pH of the control product is adjusted to 6.7 before Iyophilization. In this contlol product formulation, there is no means disclosed for maintaining the pH
of the control product at 6.7 after rehydration. Variability in pH of the con$rol product after reforrnulation can lead to inconsistent results. Additionally, there is no suggestion that the disclosed control product formulation would stabilize immunoreactivity of CK or CK isoenzymes.
There is a need for a formulation for stabilizing enzymatic activity and immunore~ctivity of CK and CK
isoenzymes which can be used to stabilize h-~man serum samples containing CK and CK isoenzymes and as a reconstitutable lyophilized CK and CK isoenzyme control product which exhibits little temperature of hydration effect and which is stable after reconstitution.
D~S~L~ lN~Nl~ON
The formulation of this invention for stabilizing 2 5 enzymatic activity and immunoreactivity of creatine kinase or creatine kinase isoenzymes comprising:
(a) animal serum;
~b) creatine kinase and/or creatine kinase isoenzymes;
3 0 (c) an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7 0.1;
(d) an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or crea~ine kinase 3 5 isoenzyme thiol groups;
, ` . 2 ~ 7 (e~ an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatin~
kinase isoenzymes; and (f) an effective amount of an antimicrobial to 5 prevent microbial growth in the ~ormulation.
The Iormulation oE ~he in~stant invention can be used to s~abilize enzymatic activity and ;mmunoreactivity of CK and CK
isoenzymes in human serum samples and in reconstitutable lyophilized control products for the assay of CK or CK-MB in 10 human serum. When used as a reconstitutable lyophilized control product, the formulation of the instant invention exhibits little temperature of hydration effect providing a cvntrol product in which enzymatic activity and immunoreactivi~y remain constant after reconstitution for at 15 least two weeks at 4C.
D:EiSC~le~
The formulation of the instant invention is useful for stabilizing both the enzymatic activity and immunoreactivity of CK and CK isoenzymes in human serum samples and in 2 0 recons~ituted Iyophilized CK and CK isoenzyme control products.
The formulation can take the form of an additive to blood collection tubes for the collection of human serum samples to be assayed for the dete~mination of CK and CK isoenzyme levels or ~he formula~ion can ~ake the form of a reconstitutable 25 lyophilized control product to be used in an assay for the determination of CK andlor CK isoenzyme levels.
The formulation of the instant invention is serum-based.
Where the formulation is used in blood collection tubes to preserve the stability of CK and CK isoenzymes in human serum 3 0 samples, the serum is that collected as the sample. Where the formulation talces the form of a reconstitutable Iyophilized CK
and/or CK ;soenzyme control product, the serum can be any animal serum. Horse serum is preferred.
When the formulation is used as a control product, i~ is 3 5 preferred that the serum have substantially low or no residual "
}~ 6 2 1 ~
CK and CK isoenzyme enzymatic activity or immunoreactivity.
~, Residual enzymatic activity and immunoreactivity can be removed from the serum by heat treatment of the serum. The serum can be heated to any temperature for a length of time S which will denature the CK or CK isoenzyme, such as S6C for one hour or 60C for one minute. Heat treatment of the serum for one minute at 60C is preferred.
CK and CK isoenzymes can be ~ound in the human serum samples collected for assaying for CK and CK isoenzymes or, if 10 the formulation is used as a reconstitutable lyophilized control product, CK and CK isoenzymes c~n be added to the serum base.
If the serum base of the formulation is treated to destroy residual CK and CK isoenzyme enzymatic activity and irnmunoreactivity, the CK and CK isoenzyme should be added to 15 the serum base after such treatment has occurred. The reconstitutable control product can be a control product for the assay of both CK and CK isoen~ymes, in which case, both (:K and CK isoenzymes would be present in the formulation Alternatively, the control product can be used for the assay of 2 0 only CK or a CK isoenzyme, in which case, only CK or the CK
isoenzyme would be present in the formulation. CK or CK
isoenzyme from any animal muscle or heart tissue, such as cynamolgus monkey, rabbi~ and human, can be used when the con~rol product is to be used in conjunction with a diagnostic 2 5 assay which measures the enzymatic activi~y of the CK enzyme or isoenzyme When the diagnostic assay is an immunochemical assay, animal CK or CK isoenzyme which cross-reacts with antibody to human CK or CK isoenzyme can be used Purified human CK or CK isoenzyme is preferred, 30 regardless of the type of diagnostic assay with which the control product is to be used.
One of the key elements of the formulation of the instant invention is that the formulation pH is maintained at 6.7 i 0.1, even after reconstitution of the Iyophilized control product.
3 5 Maintenance of the formulatioll pHI is achieved by including a 7 ~
2~6~7 t buffer having a pKa in the range of 6.2-7.6. Suitable buffers include piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-2-acetamidoiminodiacetic acid (ADA), 1,3-bis-[~ris(hydroxymethyl)methylamino] propane (BIS-TRIS
S PROPANE), N-2-acetamido-2-aminoethanesulfonic acid (ACES), imidazole, diethylmalonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acld (HEPES), and phosphate. PIPES buffer is preferred. An effective amount of buffer is the concentration of buffer which is needed to maintain pH of the formulation 10 between 6.6 and 6.8. A PIPES bufiFer concentration of 80-95 mM has been shown to be effectiYe.
' As described above, in order to stabilize CK and CK
isoenzyme enzymatic activity and immunoreactivity, it is important to prevent oxidation of the CK and CK isoenyzme 15 thiol groups. The formulation of the instant invention does so by containing a non-thiol antioxidant, such as ascorbic acid, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Ascorbic acid is preferred. An effective amount of non--thiol antioxidant is the concentration of antioxidant needed to 2 0 prevent oxidation of the thiol groups. Concentrations of ascorbic acid in the range of 10-100 mM have been shown to be effective, 30 mM being preferred Surprisingly, it was found that inclusion of a non-reducing polyol into the formulation conferred greater stability 2~ of CK and CK isoenzyme immunoreactivity. Any non-reducing polyol, such as sucrose, glycerol3 trehalose and mannitol, can be used in the formulation of the instant invention. Sucrose is preferred. An effective amount of non-reducing polyol is the concentration of polyol which is needed to stabilize ~he 3 0 immunoreactivity of CK and CK isoenzyme. A concentration of non-reducing polyol of 1% or greater has been ~ollnd to be effective.
Microbial growth can produce pro~eases and other enzymes which can destroy both enzymatic and antigenic 3 5 activity of CK and CK isoenzymes. Therefore, an antimicrobial 8 2 ~
îs included ;n the formulation of the instant invention to prevent microbial growth. Any aminoglycoside antimicrobial, such as gentamicin, streptomycin, neomycin and kanamycin, can be used. &entamicin is preferred. An effective amount of S aDtimicrobial is the concentration of antimicrobial needed to prevent microbial growth. A 0.1% concentration of gentamicin has been found to be effective.
The formulation of the instant invention can be used to stabilize human selum samples collected in blood collection tubes. Buffer, non-thiol antioxidant, non-reducing polyol and antimicrobial can be added in dry form to blood collection tubes for use in collec~ing the human serum samples containing CK and CK isoenzymes.
Alternatively, the formulation can be used as a l S reconstitutable lyophilized control product. All of the components, except CK and CK isoenzyme, can be added in any order to the serum base of the formulation. The formulation can then be pH adjusted to 6.7i0.05, sterile filtered and heat ~eated, if so desired. CK and CK isoenzyme must be added after pH adjustment and heat treatment so as to avoid destruction of CK and CK isoenzyme. The serum-based formulation can then be lyophilized by any standard Iyophilization technique. Since the lyophilized control product is stable, it can be reconstituted with water and used in - 2 5 conjunction with, for example, automated clinical analyzers - utilized for the measurement of CK and/or (:K isoenzymes.
Once reconstituted, the formulation of the instant inverltion has been found to remain stable for at least two weeks when stored at 4C.
The following examples illustrate the invention.
~-- 9 EiXAMPLES
E~AMPLE I - PREPAR~TIQ~ OF lRECQN~T[IUT~BLE
s L~ P~E2 MB LD~R~A~
I. Preparation of CK-MB Calibrator Matrix - To 23 liters of horse serum (Pel-freez Biologicals) was 10 added 616.4 gm of 80 mM PIPES 1.5 Na salt (Sigma Corp.~, 23 gm of gentamicin sulfate (Sigma Corp.3, 230 gm of sucrose ~Ul~a Pure) and 121.6 gm ascorbic acid (Calbiochem Corp.) with continuous mixing until all additives were completely dissolved in the horse serum. While stirring vigorously, but 15 not so as to cause foaming, the horse serum mixture was adjusted to a pH of 6.7iO.05 at room temperature by slowly adding sodium hydroxide.
After pH adjustment, the horse serum mixture was filtered through a 0.2-mieron filter into a heat treatment tank 20 containing a submersible heating coil. The horse serum mixture was heat treated to remove residual CK and CK
isoenzyme by heating the mixture to SO~C for one minute.
Horse serum containing ~he above-identified additives, adjusted to pH 6.7iO.05, filtered and heat treated is CK-MB
25 Calibrator Matrix. The Matrix was eovered and stored refrigerated for further use.
II. Preparation of CK-MB Stock Solution A lyophilized preparation of CK-MB (Lee Scientific~ was 3 0 used as the source of CK isoenzyme. The lyophilized S:K-MB
preparation was rehydra~ed with 16 mL of (: alibrator Matrix to prepare CK-MB S~ock Solution. To three lO0 mL volumetric flasks was added 25 ~,IL of Stock Solution and enough Matrix to bring the resulting solution to 100 mL. These solutions were 35 used as samples on the acat~plws immunoassay system (E~. I. du Pont de Nemours and Company) and analyzed for mass CK-MB
content using a mass immunochemical assay. Results of this assay showed that the CK-MB S~ock Solution had a CK-MB
concentra~ion of 383,000 ,ug/L
S III. Formulation of CK-MB Calibrator Levels 1, 2, Low, Medium, High and 3 Using the CK-MB concentration of CK-MB Stock Solution determined in Section II., above, it was calculated, using a mass balance equation, that 9.9 gm of (:K-MB Stock Solution 10 needed to be added ~o 9070 gm of A~atrix to bring the Matrix to a target CK-MB concentration of 420 ~,lg/I,. The Matrix was assayed on the aca~plus immunoassay system to determine CK-MB concentration. 'rhe result was lower ~han the expected 420 llg/L and an additional 2.3 gm of CK-MB Stoclc Solution 15 was added to the Ma~ix. This was de3ignated Calibrator Level 3. Calibrator Matrix alone was used as Calibrator Level 1, containing 0 llg/mL CK-MB. Calibrator Levels High (200 ~glmL), Medium (lOO,ug/mL), Low (50 ~lg/mL) and 2 (20 ,ug/mL) were prepared by mixing together 1414 gm Matrix 20 and 1286 gm Level 3, 2057 gm Ma~rix arld 643 gm Level 3, 2379 gm Ma~rix and 321 gm Level 3 and 6200 gm Matrix with 310 gm Level 3, respectively.
For each level of CK-MB Calibrator, Iyophilization vials were filled with 5.39 gm of calibrator and topped with half-5 inserted slotted s~oppers. The vials were Iyophilized accordingto a routine protocol. The vials were frozen to apprcximately -38C. When the condenser reached approximately -80C, the chamber was evacuated to 200 millitorr and the shelf tempera~ure was gradually increased to 30C over 30 approximately 45 hours. The chamber was then held at 30C
and 30 millitolT for ten hours. The Iyophilized vials were stoppered under a vacuum of approxima~ely 30 millitorr and cooled to 4C.
2 ~
EXA~MPLE II - ~TAlBlI,Il~ ~;; OF RE~C)NSTIT5~ATED CK-MB
3RATC~RS
One vial each of lyophilized CK-MB Calibrator Levels 1, 2, Low, Medium, High and 3, prel)ared as described ;n Example I, 5 were reconstituted with 5.0 mI, of room temperature water on each of Days 0, 8, 22 and 44. The reconstituted vials prepared on Days 1, 8 and 22 were storecl at 4C until Day 44. Cal;brator reconstituted on Day 44 served as a control as it contained the original amount of CK-MB in each calibrator level. On Day 44, 10 three replicates of each level of reconstituted CK-MB Calibrator from each day of reconsti~ution were assayed on the aca~plus immunoassay system to determine the amount of CK-MB in each CK-MB calibrator level using a mass immunochemisal CK-MB method of determination. M[eans of the replicates, standard 15 deviations and percent coefficients of variation were calculated.
Using the slope and intercept calculated ~rom a linear least squares correlation for each level, a percent loss per day was calculated. Prom the percent loss per day was calcula~ed the number of days from reconstitution undl a 5% loss of CK-MB
2 0 would result for each reconstituted calibrator level. Results are depicted in Table 1, below.
_ l 2 2 ~ 7 ~` ~G9~]3LE 1 i DAYS FROM MEAN NU~IBEROF
RECONSTTrL~ON CK-~EI% LOSS DAYS FOR
5 LE~3L 5~EEIlESI~i 31 LkL~aC,y. P~R DAY 5% LOSS
1 0 3q.15 1.29 3.79 1 B 34.03 0.84 2.46 1 22 34.67 0.41 1.17 1 0 1 44 34.75 1.13 3.25 i 0 63.28 0.2S 0.39 0.06 77 2 8 64.02 0.93 1.45 2 22 65.07 0.96 1.47 1 5 2 44 64.18 0.10 0.16 !
LOW 0 112.07 0.70 Q.63 0.15 34 ~OW 8 1~0.07 2.85 2.59 LOW 22 115.23 4.22 3.66 2 0 LOW 44 116.10 3.90 3.36 ~DEDIUM 0 192.35 4.17 2.17 0.22 23 ~DEDIUM 8 202.40 5.09 2.52 MEDIlUM 22 207.07 6.49 3.14 2 5 MEDIUM 44 211.23 3.85 1.82 HIGH 0 388.95 9.97 2.S6 0.19 26 H~ 8 396.63 1.76 0.44 H~GH 22 406.80 2.26 0.56 3 0 HIGH 44 421.65 11.95 2.83 3 0 850.93 5.39 0.63 0.08 64 3 8 888.40 12.30 1.38 3 22 8~0.65 g.69 1.10 3 ~ 3 44 891.5S 3.18 0.3fi .
Results shown in Table 1, above, indicate that the reconstitutable lyophilized CK-MB CalibTator of the instant invention will not lose more than 5% of the original amount of 5 CK-MB contained in the calibrator for at leas~ 23 days after reconstitution when stored at 4~C.
EXAMPLE III - l~e9F ~IYDRAI'IO~ EFFEÇ E
Three vials for each of Levels 2 and 3 of the CK-MB
Calibrato~ of the instan~ invention, prepared according to Example I, above, were rehydrated wi~h water; one vial for each of water temperatures of 4, 25 and 37C. Also rehydrated at 4, ~5 and 37C were samples of lyophilized 15 stripped human serum at low and high levels of CK-MB, prepared according to U. S. Patent No. 4,264,471, issued to Briggs on April 28, 1981, and commerical CK-MB calibrators and controls from the Tandem~-E CKMB II ImmunoEnzMetric Assay ~Hybritech, Inc.), the Stratus~) CK-MB Fluorometric 20 Enzyme Immunoassay Kit (Baxter Diagnostics, Inc., Dade Division~ and Cala. The reconstituted calibrators and controls were assayed in triplicate for the amount of CK-MB contained therein using the aca@~plus immunoassay system and a mass immunochemical method of determination. Mean CK-MB
2 5 values were determined for each sample and the percent change in CK-MB value fiom that of the 4C rehydrated sample was calculated: Results are depicted in Table 2, below.
i. 1 4 --A~i 2 1 ~ 6 I ~ 7 `#
L~ 2 ~:k~ % LOS~ OF CK-MB
SAM~ ~ 2 ~ 37 INVENTION, LEVEL 2 1~.2 14.9 13.8 2.0 9.2 INVENTION, LE~VEL 3 340.0 324.0 318.0 4.7 6.5 STRIPPED HUMAN
SERllM, LOW 14.6 10.9 8.0 2S.3 4~.2 STRIPPED HUMAN
SERUM, HIGH ~68.0 222.0 170.0 17.2 36.6 1 5 TANDEM~)-E CONTROL, LOW 15.1 14.0 14.1 7.3 6.6 TANDEM~)-E CONT~OL, HIGH 56.3 52.7 50.8 6.4 9.8 2 0 TANDEM~)-E CALIBRATOR, HI&H 35.7 36.3 33.3 -1.7 6.7 STRATUS~) CALIBRATOR, MID 16.9 13.4 10.4 20.7 38.5 g STRATUS~9 CALIBRATOR, HIGH 37.9 30.0 21.7 20.8 42.7 CALA, PSID 25.8 25.3 25.1 1.9 2.7 3 0 CALA,HIGH 60.4 58.9 57.4 2.5 5.0 As can be seen from Table 2, the reconstitutable lyophilized CK-MB control product of the instant invention exhibit little temperature of hydration effect. It is desirable 3 ~ that the ternperature of hydration effect be as small as possible Only the Cala controls exhibited lower temperature of hydration efPect than did the calibrators of the instant invention. The Tandem~)-E calibrator and controls performed ~- connparably to the instant invention at 37C, but worse at 25C.The Stratus(~) and stripped hunnan serum calibrators showed significantly greater temperature of hydration effect than did the reconstitutable lyophilized CK-MB control product of the S instant invention. The results i~or stripped serum show the magnitude of temperature of rehydration effect one could expect without the instant invention.
Claims (10)
1. A formulation for stabilizing enzymatic activity and immunoreactivity of creatine kinase or creatine kinase isoenzymes comprising:
(a) animal serum;
(b) creatine kinase and/or creatine kinase isoenzymes;
(c) an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7?0.1;
(d) an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or creatine kinase isoenzyme thiol groups;
(e) an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatine kinase isoenzymes; and (f) an effective amount of an antimicrobial to prevent microbial growth in the formulation.
(a) animal serum;
(b) creatine kinase and/or creatine kinase isoenzymes;
(c) an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7?0.1;
(d) an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or creatine kinase isoenzyme thiol groups;
(e) an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatine kinase isoenzymes; and (f) an effective amount of an antimicrobial to prevent microbial growth in the formulation.
2. The formulation of claim 1 wherein the buffer is selected from the group consisting of piperazine-N,N'-bis(2-ethanesulfonic acid), N-2-acetamidoiminodiacetic acid, 1,3-bis-[tris(hydroxymethyl)methylamino] propane, N-2-acetamido-2-aminoethanesulfonic acid, imidazole, diethylmalonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and phosphate.
3. The formulation of claim 1 wherein the non-thiol antioxidant is selected from the group consisting of ascorbic acid, butylated hydroxyanisole and butylated hydroxytoluene.
4. The formulation of claim 1 wherein the non-reducing polyol is selected from the group consisting of sucrose, glycerol, trehalose and mannitol.
5. The formulation of claim 1 wherein the buffer is piperazine-N,N'-bis-2-ethanesulfonic acid), the non-thiol antioxidant is ascorbic acid, the non-reducing polyol is sucrose and the antimicrobial is gentamicin.
6. A stable water reconstitutable lyophilized control product for the assay of creatine kinase or creatine kinase isoenzymes comprising:
(a) animal serum having low residual levels of creatine kinase or creatine kinase isoenzyme enzymatic activity and immunoreactivity;
(b) creatine kinase and/or creatine kinase isoenzyme;
(c) an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7?0.1;
(d) an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or creatine kinase isoenzyme thiol groups;
(e) an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatine kinase isoenzymes; and (f) an effective amount of an antimicrobial to prevent microbial growth in the formulation.
(a) animal serum having low residual levels of creatine kinase or creatine kinase isoenzyme enzymatic activity and immunoreactivity;
(b) creatine kinase and/or creatine kinase isoenzyme;
(c) an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7?0.1;
(d) an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or creatine kinase isoenzyme thiol groups;
(e) an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatine kinase isoenzymes; and (f) an effective amount of an antimicrobial to prevent microbial growth in the formulation.
7. The water reconstitutable lyophilized control product of claim 6 wherein the animal serum is heat-treated horse serum.
8. The water reconstitutable lyophilized control product of claim 7 wherein the buffer is piperazine-N,N'-bis(2-ethanesulfonic acid), the non-thiol antioxidant is ascorbic acid, the non-reducing polyol is sucrose and the antimicrobial is gentamicin.
9. A blood collection tube additive for stabilizing enzymatic activity and immunoreactivity of creatine kinase or creatine kinase isoenzymes contained in human serum samples comprising:
(a) an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7?0.1;
(b) an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or creatine kinase isoenzyme thiol groups;
(c) an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatine kinase isoenzymes; and (d) an effective amount of an antimicrobial to prevent microbial growth in the formulation.
(a) an effective amount of a buffer having a pKa in the range of 6.2-7.6 to maintain pH of the formulation at 6.7?0.1;
(b) an effective amount of a non-thiol antioxidant to prevent oxidation of creatine kinase or creatine kinase isoenzyme thiol groups;
(c) an effective amount of a non-reducing polyol to stabilize immunoreactivity of creatine kinase or creatine kinase isoenzymes; and (d) an effective amount of an antimicrobial to prevent microbial growth in the formulation.
10. The blood collection tube additive of claim 9 wherein the buffer is piperazine-N,N'-bis(2-ethanesulfonic acid), the non-thiol antioxidant is ascorbic acid, the non-reducing polyol is sucrose and the antimicrobial is gentamicin.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/939,861 | 1992-09-14 | ||
| US07/939,861 US5298406A (en) | 1992-09-14 | 1992-09-14 | Formulation for stabilizing enzymatic activity and immunoreactivity of creatine kinase and creatine kinase isoenzymes |
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|---|---|
| CA2106167A1 true CA2106167A1 (en) | 1994-03-15 |
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| CA002106167A Abandoned CA2106167A1 (en) | 1992-09-14 | 1993-09-14 | Formulation for stabilizing enzymatic activity and immunoreactivity of creatine kinase and creatine kinase isoenzymes |
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| Country | Link |
|---|---|
| US (1) | US5298406A (en) |
| EP (1) | EP0596218B1 (en) |
| JP (1) | JP2930277B2 (en) |
| CA (1) | CA2106167A1 (en) |
| DE (1) | DE69319693T2 (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU692547B2 (en) * | 1994-05-06 | 1998-06-11 | Beckman Instruments, Inc. | Liquid stable thiol activator |
| JP3625503B2 (en) | 1994-11-11 | 2005-03-02 | 旭化成ファーマ株式会社 | Composition-containing composition for clinical testing |
| WO1997008551A1 (en) * | 1995-08-31 | 1997-03-06 | First Medical, Inc. | Methods and antibodies for detecting creatine kinase |
| WO1998040749A1 (en) * | 1997-03-12 | 1998-09-17 | Yamasa Corporation | Method for detecting vasculopathy |
| US5998158A (en) * | 1997-05-14 | 1999-12-07 | Fuji Photo Film Co., Ltd. | Glucose free, stable dry analytical element for quantitative analysis of creatine kinase MB isozyme |
| JP3750955B2 (en) * | 1997-04-18 | 2006-03-01 | 富士写真フイルム株式会社 | Dry analytical element for quantification of creatine kinase or its MB isozyme |
| DE19755079A1 (en) | 1997-12-11 | 1999-06-17 | Roche Diagnostics Gmbh | Stabilized reagent and method for the determination of creatine kinase |
| US5908864A (en) * | 1998-05-28 | 1999-06-01 | Dymatize Enterprises | Creatine gel |
| JP2000154200A (en) * | 1998-11-20 | 2000-06-06 | Kanegafuchi Chem Ind Co Ltd | Immobilization of compound |
| JP2001299361A (en) * | 2000-04-28 | 2001-10-30 | Oriental Yeast Co Ltd | Recombinant human creatine kinase heterodimer with solution stability |
| WO2003097237A2 (en) * | 2002-05-13 | 2003-11-27 | Becton, Dickinson, And Company | Protease inhibitor sample collection system |
| EP1435241A1 (en) * | 2002-12-30 | 2004-07-07 | Kyokuto Pharmaceutical Industrial Co. Ltd | Method for stabilizing blood, serum, or plasma specimen and container containing ph buffer agent |
| CN105974117B (en) * | 2016-05-04 | 2017-09-01 | 同昕生物技术（北京）有限公司 | CK MB calibration objects dilutions and its application in clinical CK detections |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1102225A (en) * | 1976-09-13 | 1981-06-02 | Ivan E. Modrovich | Stabilized liquid enzyme and coenzyme compositions and method of preparing same |
| DE2831390C2 (en) * | 1978-07-17 | 1980-08-14 | Boehringer Mannheim Gmbh, 6800 Mannheim | Control serum for clinical-chemical analysis with a certain content of creatine kinase |
| US4325832A (en) * | 1979-03-05 | 1982-04-20 | Beckman Instruments, Inc. | Enzyme reference composition |
| JPS5653465A (en) * | 1979-10-05 | 1981-05-13 | Toyo Jozo Co Ltd | Improved method in measurement of enzyme immunity |
| EP0044532B1 (en) * | 1980-07-18 | 1986-04-23 | E.I. Du Pont De Nemours And Company | Monothioglycerol as thiol-protector in lyophilized materials |
| US4339533A (en) * | 1980-07-30 | 1982-07-13 | Technicon Instruments Corporation | Stabilization of creatine kinase (CK) and its application as a reference standard |
| US4442212A (en) * | 1980-11-10 | 1984-04-10 | E. I. Du Pont De Nemours & Company | Monothioglycerol as thiol-protector in lyophilized materials |
| DE3540076A1 (en) * | 1985-11-12 | 1987-05-14 | Boehringer Mannheim Gmbh | METHOD FOR STABILIZING CREATINE KINASE |
| JPS62228272A (en) * | 1986-03-27 | 1987-10-07 | Amano Pharmaceut Co Ltd | Stable peroxidase formulation |
| US4994375A (en) * | 1988-07-11 | 1991-02-19 | Baxter International Inc. | Stable human serum based control and/or calibrant |
| US5102788A (en) * | 1988-11-21 | 1992-04-07 | Hygeia Sciences, Inc. | Immunoassay including lyophilized reactant mixture |
| GB9010742D0 (en) * | 1990-05-14 | 1990-07-04 | Quadrant Bioresources Ltd | Stabilization of biological macromolecular substances |
-
1992
- 1992-09-14 US US07/939,861 patent/US5298406A/en not_active Expired - Fee Related
-
1993
- 1993-09-09 DE DE69319693T patent/DE69319693T2/en not_active Expired - Fee Related
- 1993-09-09 EP EP93114468A patent/EP0596218B1/en not_active Expired - Lifetime
- 1993-09-14 JP JP5228586A patent/JP2930277B2/en not_active Expired - Lifetime
- 1993-09-14 CA CA002106167A patent/CA2106167A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0596218A2 (en) | 1994-05-11 |
| JPH06189760A (en) | 1994-07-12 |
| JP2930277B2 (en) | 1999-08-03 |
| EP0596218B1 (en) | 1998-07-15 |
| EP0596218A3 (en) | 1994-06-01 |
| DE69319693T2 (en) | 1998-12-10 |
| DE69319693D1 (en) | 1998-08-20 |
| US5298406A (en) | 1994-03-29 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |